Kathleen Haugk

Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.

Overexpression of the type-1 insulin-like growth factor receptor increases ligand-dependent proliferation and differentiation in bovine skeletal myogenic cultures

Previous studies demonstrated that overexpression of the type‐1 insulin‐like growth factor (IGF) receptor (IGF‐1R) in skeletal myogenic cell lines increased proliferation and differentiation responses to IGF. However, it was unclear if such manipulations in primary, untransformed skeletal myogenic cells would result in modulation of these responses, which may be more stringently regulated in primary cells than in myogenic cell lines. In this study, low passage untransformed fetal bovine myogenic cultures were infected with a replication‐deficient retroviral expression vector (LISN) coding for the human IGF‐1R or with a control retroviral vector (LNL6). Bovine myogenic cultures infected with the LISN vector (Bov‐LISN) displayed ten times more IGF‐1Rs than controls (Bov‐LNL6). Bov‐LISN myogenic cultures exhibited elevated rates of IGF‐I‐stimulated proliferation and increased rates of terminal differentiation which were reduced to control levels by the anti‐human IGF‐1R antibody αIR3. These findings indicate overexpression of the IGF‐1R can enhance IGF sensitivity and thereby modify the proliferation and differentiation behavior of untransformed low passage myoblasts. Such manipulations may be useful to increase muscle mass in clinical or agricultural applications.

Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF1

Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1–...

Response of the Insulin-Like Growth Factor (IGF) System to IGF-IR Inhibition and Androgen Deprivation in a Neoadjuvant Prostate Cancer Trial: Effects of Obesity and Androgen Deprivation

CONTEXT Prostate cancer patients at increased risk for relapse after prostatectomy were treated in a neoadjuvant study with androgen deprivation therapy (ADT) in combination with cixutumumab, an inhibitory fully human monoclonal antibody against IGF receptor 1 (IGF-IR). OBJECTIVE A clinical trial with prospective collection of serum and tissue was designed to test the potential clinical efficacy of neoadjuvant IGF-IR blockade combined with ADT in these patients. The effect of body mass index (BMI) on response of IGF-IR/insulin components to IGF-IR blockade was also examined. DESIGN Eligibility for the trial required the presence of high-risk prostate adenocarcinoma. Treatment consisted of bicalutamide, goserelin, and cixutumumab for 13 weeks before prostatectomy. Here we report on an analysis of serum samples from 29 enrolled patients. Changes in IGF and glucose homeostasis pathways were compared to control samples from patients in a concurrent clinical trial of neoadjuvant ADT alone. RESULTS Significant increases were seen in GH (P = .001), IGF-I (P < .0001), IGF-II (P = .003), IGF binding protein (IGFBP)-3 (P < .0001), C-peptide (P = .0038), and insulin (P = .05) compared to patients treated with ADT alone. IGFBP-1 levels were significantly lower in the cixutumumab plus ADT cohort (P = .001). No significant changes in blood glucose were evident. Patients with BMIs in the normal range had significantly higher GH (P < .05) and IGFBP-1 (P < 0.5) levels compared to overweight and obese patients. CONCLUSIONS Patients with IGF-IR blockade in combination with ADT demonstrated significant changes in IGF and glucose homeostasis pathway factors compared to patients receiving ADT alone. In the patients receiving combination therapy, patients with normal BMI had serum levels of glucose homeostasis components similar to individuals in the ADT-alone cohort, whereas patients with overweight and obese BMIs had serum levels that differed from the ADT cohort.

Transforming Growth Factor-β–Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition

Purpose: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. Results: In castrate mice, the tumors were delayed in G2 with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G1 or G2 delay. Transforming growth factor-β–stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G1. When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. Conclusions: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634–41)

A phase I study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer

Background Niclosamide, an FDA-approved anti-helminthic drug, has activity in preclinical models of castration-resistant prostate cancer (CRPC). Potential mechanisms of action include degrading constitutively active androgen receptor splice variants (AR-Vs) or inhibiting other drug-resistance pathways (e.g., Wnt-signaling). Published pharmacokinetics data suggests that niclosamide has poor oral bioavailability, potentially limiting its use as a cancer drug. Therefore, we launched a Phase I study testing oral niclosamide in combination with enzalutamide, for longer and at higher doses than those used to treat helminthic infections. Methods We conducted a Phase I dose-escalation study testing oral niclosamide plus standard-dose enzalutamide in men with metastatic CRPC previously treated with abiraterone. Niclosamide was given three-times-daily (TID) at the following dose-levels: 500, 1000 or 1500mg. The primary objective was to assess safety. Secondary objectives, included measuring AR-V expression from circulating tumor cells (CTCs) using the AdnaTest assay, evaluating PSA changes and determining niclosamide’s pharmacokinetic profile. Results 20 patients screened and 5 enrolled after passing all screening procedures. 13(65%) patients had detectable CTCs, but only one was AR-V+. There were no dose-limiting toxicities (DLTs) in 3 patients on the 500mg TID cohort; however, both (N = 2) subjects on the 1000mg TID cohort experienced DLTs (prolonged grade 3 nausea, vomiting, diarrhea; and colitis). The maximum plasma concentration ranged from 35.7–82 ng/mL and was not consistently above the minimum effective concentration in preclinical studies. There were no PSA declines in any enrolled subject. Because plasma concentrations at the maximum tolerated dose (500mg TID) were not consistently above the expected therapeutic threshold, the Data Safety Monitoring Board closed the study for futility. Conclusions Oral niclosamide could not be escalated above 500mg TID, and plasma concentrations were not consistently above the threshold shown to inhibit growth in CRPC models. Oral niclosamide is not a viable compound for repurposing as a CRPC treatment. Clinical trial registry Clinicaltrials.gov: NCT02532114

Abstract 2324: Phospho-MED1 mediates the transcriptional regulation of AR splice variants in castration-resistant prostate cancer

The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). Prior to treatment with enzalutamide or abiraterone, around 20-30% of CRPC patients already have high levels of AR-Vs in their metastases, especially AR v567es , and this portends a rapid progression and shorter survival. However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation still remains unclear. A distinct transcriptome enriched with cell cycle genes (like UBE2C) has been associated with AR-Vs, which indicates the possibility of a different transcriptional mechanism than that of wild type AR. In our study, we performed co-immunoprecipitation, ChIP and luciferase reporter assays to explore the components involved in AR v567es activated UBE2C transcription. We found that (i) MED1 is necessary for AR v567es induced UBE2C up-regulation and subsequent prostate cancer cell growth; (ii) p-MED1 is recruited to AR v567es independent of full-length AR; (iii) p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of AR v567es , (iv) AR v567es enhanced UBE2C transcription could be blocked by silencing MED1; (v) AR v567es /p-MED1 signaling cross talks with the PI3K-AKT pathway but not the MAPK pathway, and (vi) FoxA1 and histone methylation are involved in AR v567es /p-MED1 induced UBE2C long range chromatin interactions. In summary, these data indicate that p-MED1 serves as a key mediator in AR v567es transcriptional regulation and suggests a mechanism by which AR-Vs promote the development and progression of CRPC. Citation Format: Gang Liu, Cynthia Sprenger, Pin-Jou Wu, Shihua Sun, Takuma Uo, Kathleen Haugk, Kathryn Soriano Epilepsia, Stephen Plymate. Phospho-MED1 mediates the transcriptional regulation of AR splice variants in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2324. doi:10.1158/1538-7445.AM2014-2324

Abstract 2114: UGT2B17 is a unique co-regulator of the androgen receptor and androgen receptor splice variants in prostate cancer

Background: Expression of the steroid glucuronidating enzyme, UGT2B17, is suppressed by ligand bound full-length androgen receptor (AR-FL). However, we have shown that the AR constitutively active AR splice variants AR-V7 and ARv567es significantly increase the expression of UGT2B17 in prostate cancer cell lines and xenografts. Further, recent studies demonstrated that UGT2B17 is increased in patients with castrate recent prostate cancer. We have also shown that UGT2B17 is increased in multiple cell lines when AR splice variants (AR-Vs) increase in response to androgen deprivation therapies such as enzalutamide and abiraterone. Objective: Since UGT2B17 functions to glucuronidate dihydrotestosterone (DHT) and decrease intracellular androgen, it would be a potential mechanism for generation of constitutively active AR-Vs by androgen deprivation therapy-induced alternative splicing. In xenografts that have been shown to harbor genomic rearrangements that encode for AR-Vs, we see marked elevations of UGT2B17, an indication that it was not necessary for generation of AR-Vs but has additional activities to enhance AR-Vs signaling. The purpose of this study was to determine the interaction of UGT2B17 with AR-Vs, in cell lines that have not been shown to harbor AR genomic rearrangements. Results: In the parental LNCaP cell line, expression of UGT2B17 decreased after growth in charcoal stripped serum (CSS) supplemented with 10-9M DHT. Expression increased following growth in CSS alone and when enzalutamide was added to CSS. In contrast, in LNCaP-abl or LNCaP 95 cells, which are castration resistant sublines that express AR-Vs, basal UGT2B17 was significantly elevated compared with LNCaP baseline levels. Further, in these castrate resistant sublines, UGT2B17 exhibited less suppression by DHT and there was no increase in UGTB17 over baseline following enzalutamide treatment. ChIP assays in parental LNCaP cells stably transfected with ARv567es demonstrated binding to the UGT2B17 promoter in the absence of DHT and decreased binding in the presence of DHT. In control LNCaP cells transfected with empty vector, no binding to the UGT2B17 promoter was seen in the presence of DHT. Similar results were seen in enzalutamide resistant VCaP xenografts: UGT2B17 was elevated in the castrated state but suppressed when DHT was added. When lysates from LNCaP-abl, LNCaP 95, or VCaP cells grown in CSS were immunoprecipitated with UGT2B17 antibody then blotted with an AR-N terminal antibody, AR and AR-Vs were noted to be present. AR-negative M12 cells and LNCaP cells grown in DHT demonstrated no pull-down of AR in the UGT2B17 IP9s. Interpretation: This study demonstrated that UGT2B17 is a unique co-regulator of AR-Vs activity and it is up-regulated by AR-Vs and suppressed by androgen transactivated AR-FL receptor. UGT2B17 may thus be part of an autocrine-loop driving castrate resistant prostate cancer by AR-Vs. Citation Format: Gang Liu, Shihua Sun, Kathleen Haugk, Cynthia Sprenger, Xeusen Dong, Elahe Mostaghel, Stephen R. Plymate. UGT2B17 is a unique co-regulator of the androgen receptor and androgen receptor splice variants in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2014-2114

Abstract 631: Androgen receptor(AR) splice variants inhibit responses to IGF-IR inhibition in prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Constitutively active splice variants of the androgen receptor (AR) are frequently expressed (59% of metastases) following castration in prostate cancer patients and are associated with earlier tumor recurrence compared to tumors containing only wild-type (wt) AR. In most tumors the variant is expressed along with wt-AR. When wtAR and variant AR are expressed in cells following castration there is a marked decrease in IGF type 1 receptor expression (IGF-IR), an AR target whose expression is increased by wtAR. In order to understand these findings we expressed the AR splice variant in which exons 5, 6, and 7 are skipped during AR splicing resulting in constitutively active ARv567es along with wtAR in a prostate cancer cell line not expressing AR, M12, as well as ARv567es in AR positive LnCaP cells. In both lines ARv567es was found only in the nucleus in the absence of ligand but wtAR had also been transported to the nucleus and absolute levels of AR protein were increased in the absence of ligand in cells expressing the variant. The increase in wtAR protein is due to decreased degradation of wtAR associated with ARv567es. When ligand was added, PSA response was increased 5-fold over cells transfected only with wtAR. Further, whereas transfection with wtAR enhances IGF-IR expression and response to IGF-I ligand, in the presence of ARv567es IGF-IR expression in response to IGF-I was suppressed. When ARv567es was HA tagged and expressed along with wtAR pull-down of ARv567es with HA resulted in concomitant precipitation of wtAR. Subsequent deletion analysis of ARv567es demonstrated direct binding between the two ARs in specific cystine-rich domains. Further, wtAR-induced IGF-IR expression is a non-genomic AR cytoplasmic action through binding to src and MAPK phosphorylation. Although ARv567es binds src, phosphorylation of MAPK does not occur. Finally, when LnCaP cells expressing ARv567es were grown sc with MatrigelTM in immuno-compromised mice there was no response to castration as opposed to slowed growth of LnCaP controls. These data indicate that the presence of the castration-induced ARv567es not only effects progression of prostate cancer by its own constitutive activity but also changes the function of the wt or full-length AR and provides an additional mechanism for AR-induced prostate cancer progression following castration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 631.